Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases.

Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKß-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.

Rolipram treatment during consolidation ameliorates long-term object location memory in aged male mice.

Normal aging is accompanied by cognitive and memory impairments that negatively impact quality of life for the growing elderly population. Hippocampal function is most vulnerable to the deleterious effects of aging, and deficits in hippocampus-dependent memories are common amongst aged individuals. Moreover, signaling networks such as the cAMP/PKA/CREB pathway, which are critical for memory consolidation, are dampened in healthy aged subjects. Phosphodiesterase (PDE) enzymes that break down cAMP are also affected by aging, and increased break down of cAMP by PDEs may contribute to reduced activity of the cAMP/PKA/CREB signaling network in the brain of aged individuals. Here, we report that the PDE4 inhibitor rolipram administered during consolidation of hippocampus-dependent object location memory improves aged-related spatial memory deficits in aged mice.

Effects of Pharmacologically Targeting Neuroimmune Pathways on Alcohol Drinking in Mice Selectively Bred to Drink to Intoxication.

BACKGROUND: Rodent models of high alcohol drinking offer opportunities to better understand factors for alcohol use disorders (AUD) and test potential treatments. Selective breeding was carried out to create 2 unique High Drinking in the Dark (HDID-1, HDID-2) mouse lines that represent models of genetic risk for binge-like drinking. A number of studies have indicated that neuroimmune genes are important for regulation of alcohol drinking. We tested whether compounds shown to reduce drinking in other models also reduce alcohol intake in these unique genetic lines. METHODS: We report tests of gabapentin, tesaglitazar, fenofibrate, caffeic acid phenethyl ester (CAPE), ibrutinib, and rolipram. Although these compounds have different mechanisms of action, they have all been shown to reduce inflammatory responses. We evaluated effects of these compounds on alcohol intake. In order to facilitate comparison with previously published findings for some compounds, we employed similar schedules that were previously used for that compound. RESULTS: Gabapentin increased ethanol (EtOH) binge-like alcohol drinking in female HDID-1 and HS/NPT mice. Tesaglitazar and fenofibrate did not alter 2-bottle choice (2BC) drinking in male HDID-1 or HS/NPT mice. However, tesaglitazar had no effect on DID EtOH intake but reduced blood alcohol levels (BAL), and fenofibrate increased DID intake with no effects on BAL. CAPE had no effect on EtOH intake. Ibrutinib reduced intake in female HDID-1 in initial testing, but did not reduce intake in a second week of testing. Rolipram reduced DID intake and BALs in male and female HDID-1, HDID-2, and HS/NPT mice. CONCLUSIONS: A number of compounds shown to reduce EtOH drinking in other models, and genotypes are not effective in HDID mice or their genetically heterogeneous founders, HS/NPT. The most promising compound was the PDE4 inhibitor, rolipram. These results highlight the importance of assessing generalizability when rigorously testing compounds for therapeutic development.

Rolipram Attenuates Early Brain Injury Following Experimental Subarachnoid Hemorrhage in Rats: Possibly via Regulating the SIRT1/NF-κB Pathway.

Early brain injury (EBI) is the primary cause of poor outcome in subarachnoid hemorrhage (SAH) patients. Rolipram, a specific phosphodiesterase-4 inhibitor which is traditionally used as an anti-depressant drug, has been recently proven to exert neuroprotective effects in several central nervous system insults. However, the role of rolipram in SAH remains uncertain. The current study was aimed to investigate the role of rolipram in EBI after SAH and explore the potential mechanism. Adult male Sprague-Dawley rats were subjected to an endovascular perforation process to produce an SAH model. Rolipram was injected intraperitoneally at 2 h after SAH with a dose of 10 mg/kg. We found that rolipram significantly ameliorated brain edema and alleviated neurological dysfunction after SAH. Rolipram treatment remarkably promoted the expression of Sirtuin 1 (SIRT1) while inhibited NF-κB activation. Moreover, rolipram significantly inhibited the activation of microglia as well as down-regulated the expression of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6. In addition, rolipram increased the expression of protective cytokine IL-10. Furthermore, rolipram significantly alleviated neuronal death after SAH. In conclusion, these data suggested that rolipram exerts neuroprotective effects against EBI after SAH via suppressing neuroinflammation and reducing neuronal loss. The neuroprotective effects of rolipram were associated with regulating the SIRT1/NF-κB pathway. Rolipram could be a novel and promising therapeutic agent for SAH treatment.

Inhibition of PDE4 by low doses of rolipram induces changes in lipid and protein components of mice heart.

There is significant increasing interest in phosphodiesterase-4 (PDE4) inhibition in treatment of cardiovascular diseases. Related with this, research has focused on cellular, biochemical, molecular and structural changes in heart tissue induced by PDE4s inhibitors. However, for their clinical applicability additional studies are still needed. Fourier transform infrared spectroscopy offers promising approach to contribute such issue due to its ability in detection the changes in biomolecules. By utilizing this method, we examined the effects of PDE4 inhibition by rolipram at 0.05 mg/kg and 0.1 mg/kg doses on content of lipids and proteins, and fluidity, order and packing of membranes in naive mice heart. In treated groups, there was a significant decrease in unsaturated, saturated lipids, cholesterol esters, fatty acids, phospholipids and triacylgylcerols obtained from CH2, C=O, olefinic=CH, and COO- areas, and CH2/lipid, C=O/lipid, olefinic=CH/lipid, and COO-/lipid ratios. Additionally, olefinic=CH area and olefinic=CH/lipid ratio may suggest decreased lipid peroxidation, confirmed by thiobarbituric acid assay. Also, a higher degree of membrane order, slight increase in membrane fluidity and differences in membrane packing were obtained. Amide I and II areas and RNA/protein ratios showed that variation in protein content is not correlated with applied concentration. Analysis of amide I mode predicted alterations in secondary structures like an increase in random coils and decrease in alpha-helices. Moreover, all groups were successfully discriminated by cluster analysis. The corresponding results may help to understand the potential effects of PDE4 inhibition by rolipram.

Drug delivery to the human and mouse uterus using immunoliposomes targeted to the oxytocin receptor.

BACKGROUND: The ability to provide safe and effective pharmacotherapy during obstetric complications, such as preterm labor or postpartum hemorrhage, is hampered by the systemic toxicity of therapeutic agents leading to adverse side effects in the mother and fetus. Development of novel strategies to target tocolytic and uterotonic agents specifically to uterine myocytes would improve therapeutic efficacy while minimizing the risk of side effects. Ligand-targeted liposomes have emerged as a reliable and versatile platform for targeted drug delivery to specific cell types, tissues or organs. OBJECTIVE: Our objective was to develop a targeted drug delivery system for the uterus utilizing an immunoliposome platform targeting the oxytocin receptor. STUDY DESIGN: We conjugated liposomes to an antibody that recognizes an extracellular domain of the oxytocin receptor. We then examined the ability of oxytocin receptor-targeted liposomes to deliver contraction-blocking (nifedipine, salbutamol and rolipram) or contraction-enhancing (dofetilide) agents to strips of spontaneously contracting myometrial tissue in vitro (human and mouse). We evaluated the ability of oxytocin receptor-targeted liposomes to localize to uterine tissue in vivo, and assessed if targeted liposomes loaded with indomethacin were capable of preventing lipopolysaccharide-induced preterm birth in mice. RESULTS: Oxytocin receptor-targeted liposomes loaded with nifedipine, salbutamol or rolipram consistently abolished human myometrial contractions in vitro, while oxytocin receptor-targeted liposomes loaded with dofetilide increased contraction duration. Nontargeted control liposomes loaded with these agents had no effect. Similar results were observed in mouse uterine strips. Following in vivo administration to pregnant mice, oxytocin receptor-targeted liposomes localized specifically to the uterine horns and mammary tissue. Targeting increased localization to the uterus 7-fold. Localization was not detected in the maternal brain or fetus. Targeted and nontargeted liposomes also localized to the liver. Oxytocin receptor-targeted liposomes loaded with indomethacin were effective in reducing rates of preterm birth in mice, whereas nontargeted liposomes loaded with indomethacin had no effect. CONCLUSION: Our results demonstrate that oxytocin receptor-targeted liposomes can be used to either inhibit or enhance human uterine contractions in vitro. In vivo, the liposomes localized to the uterine tissue of pregnant mice and were effective in delivering agents for the prevention of inflammation-induced preterm labor. The potential clinical advantage of targeted liposomal drug delivery to the myometrium is reduced dose and reduced toxicity to both mother and fetus.

Development and assessment of countermeasure formulations for treatment of lung injury induced by chlorine inhalation.

Chlorine is a commonly used, reactive compound to which humans can be exposed via accidental or intentional release resulting in acute lung injury. Formulations of rolipram (a phosphodiesterase inhibitor), triptolide (a natural plant product with anti-inflammatory properties), and budesonide (a corticosteroid), either neat or in conjunction with poly(lactic:glycolic acid) (PLGA), were developed for treatment of chlorine-induced acute lung injury by intramuscular injection. Formulations were produced by spray-drying, which generated generally spherical microparticles that were suitable for intramuscular injection. Multiple parameters were varied to produce formulations with a wide range of in vitro release kinetics. Testing of selected formulations in chlorine-exposed mice demonstrated efficacy against key aspects of acute lung injury. The results show the feasibility of developing microencapsulated formulations that could be used to treat chlorine-induced acute lung injury by intramuscular injection, which represents a preferred route of administration in a mass casualty situation.

BK channel-mediated relaxation of urinary bladder smooth muscle: a novel paradigm for phosphodiesterase type 4 regulation of bladder function.

Elevation of intracellular cAMP and activation of protein kinase A (PKA) lead to activation of large conductance voltage- and Ca(2+)-activated K(+) (BK) channels, thus attenuation of detrusor smooth muscle (DSM) contractility. In this study, we investigated the mechanism by which pharmacological inhibition of cAMP-specific phosphodiesterase 4 (PDE4) with rolipram or Ro-20-1724 (C(15)H(22)N(2)O(3)) suppresses guinea pig DSM excitability and contractility. We used high-speed line-scanning confocal microscopy, ratiometric fluorescence Ca(2+) imaging, and perforated whole-cell patch-clamp techniques on freshly isolated DSM cells, along with isometric tension recordings of DSM isolated strips. Rolipram caused an increase in the frequency of Ca(2+) sparks and the spontaneous transient BK currents (TBKCs), hyperpolarized the cell membrane potential (MP), and decreased the intracellular Ca(2+) levels. Blocking BK channels with paxilline reversed the hyperpolarizing effect of rolipram and depolarized the MP back to the control levels. In the presence of H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride], a PKA inhibitor, rolipram did not cause MP hyperpolarization. Rolipram or Ro-20-1724 reduced DSM spontaneous and carbachol-induced phasic contraction amplitude, muscle force, duration, and frequency, and electrical field stimulation-induced contraction amplitude, muscle force, and tone. Paxilline recovered DSM contractility, which was suppressed by pretreatment with PDE4 inhibitors. Rolipram had reduced inhibitory effects on DSM contractility in DSM strips pretreated with paxilline. This study revealed a novel cellular mechanism whereby pharmacological inhibition of PDE4 leads to suppression of guinea pig DSM contractility by increasing the frequency of Ca(2+) sparks and the functionally coupled TBKCs, consequently hyperpolarizing DSM cell MP. Collectively, this decreases the global intracellular Ca(2+) levels and DSM contractility in a BK channel-dependent manner.

Chemopreventive effect of phosphodieasterase-4 inhibition in benzo(a)pyrene-induced murine lung cancer model.

PURPOSE: Cigarette smoking increases chronic airway inflammation, oxidative stress, and epithelial mesenchymal transition (EMT), which may result in chronic obstructive pulmonary disease (COPD) and tumor growth in the lung. Phosphodiesterase 4 (PDE4) inhibitors are known to reduce inflammation, and they have recently been introduced for the treatment of COPD. We assessed the impact of rolipram, a selective PDE4 inhibitor, on chemoprevention in benzo(a)pyrene-induced lung cancer in mice. MATERIALS AND METHODS: Female A/J mice were given a single dose of benzo(a)pyrene. Intraperitoneal administration of rolipram began 2 weeks post-carcinogen treatment and continued tri-weekly for 28 weeks. Tumor load was determined by averaging the total tumor volume in each group. RESULTS: Benzo(a)pyrene induced an average tumor size of 10.4 ± 1.7 tumors per mouse, with an average tumor load of 25.9 ± 3.8 mm(3). Rolipram significantly decreased tumor number, by 45.1%, and tumor load, by 52.9%, compared with the benzo(a)pyrene group. Ki67 staining was reduced in rolipram-treated mice compared with benzo(a)pyrene-treated mice. The increased expression of EMT markers caused by benzo(a)pyrene was inhibited by rolipram. Rolipram significantly attenuated NF-κB and Nrf2 expression in benzo(a)pyrene-induced lung cancer tissues. CONCLUSIONS: In vivo experiments in the benzo(a)pyrene-induced model of lung cancer show that PDE4 inhibition significant inhibits lung carcinogenesis. Our results provide evidence that PDE4 inhibitors may be suitable for the prevention of the lung cancer in high-risk groups, for example, heavy smokers and patients with COPD.

Adjuvant host-directed therapy with types 3 and 5 but not type 4 phosphodiesterase inhibitors shortens the duration of tuberculosis treatment.

BACKGROUND: Shortening tuberculosis treatment could significantly improve patient adherence and decrease the development of drug resistance. Phosphodiesterase inhibitors (PDE-Is) have been shown to be beneficial in animal models of tuberculosis. We assessed the impact of PDE-Is on the duration of treatment in tuberculous mice. METHODS: We analyzed the time to death in Mycobacterium tuberculosis-infected mice receiving type 4 PDE-Is (rolipram and cilomilast) and the impact on bacterial burden, time to clearance, and relapse when types 3 and 5 PDE-Is (cilostazol and sildenafil, respectively) and rolipram were added to the standard treatment. We investigated pharmacokinetic interactions between PDE-Is (cilostazol and sildenafil) and rifampin. RESULTS: The type 4 PDE-Is rolipram and cilomilast accelerated the time to death in tuberculous mice. The addition of rolipram to standard tuberculosis treatment increased bacterial burden and did not decrease the time to bacterial clearance in the lung, while the addition of the cilostazol and sildenafil reduced the time to clearance by 1 month. Cilostazol and sildenafil did not have negative pharmacokinetic interactions with rifampin. CONCLUSIONS: Type 4 PDE-Is may increase the severity of tuberculosis and should be carefully investigated for use in patients with latent or active tuberculosis. Cilostazol and sildenafil may benefit tuberculosis patients by shortening the duration of therapy.

Increased PKA signaling disrupts recognition memory and spatial memory: role in Huntington's disease.

Huntington's disease (HD) patients and mouse models show learning and memory impairment even before the onset of motor symptoms. However, the molecular events involved in this cognitive decline are still poorly understood. Here, using three different paradigms, the novel object recognition test, the T-maze spontaneous alternation task and the Morris water maze, we detected severe cognitive deficits in the R6/1 mouse model of HD before the onset of motor symptoms. When we examined the putative molecular pathways involved in these alterations, we observed hippocampal cAMP-dependent protein kinase (PKA) hyper-activation in naïve R6/1 mice compared with wild-type (WT) mice, whereas extracellular signal-regulated kinase 1/2 and calcineurin activities were not modified. Increased PKA activity resulted in hyper-phosphorylation of its substrates N-methyl-D-aspartate receptor subunit 1, Ras-guanine nucleotide releasing factor-1 and striatal-enriched protein tyrosine phosphatase, but not cAMP-responsive element binding protein or the microtubule-associated protein tau. In correlation with the over-activation of the PKA pathway, we found a down-regulation of the protein levels of some phosphodiesterase (PDE) 4 family members. Similar molecular changes were found in the hippocampus of R6/2 mice and HD patients. Furthermore, chronic treatment of WT mice with the PDE4 inhibitor rolipram up-regulated PKA activity, and induced learning and memory deficits similar to those seen in R6 mice, but had no effect on R6/1 mice cognitive impairment. Importantly, hippocampal PKA inhibition by infusion of Rp-cAMPS restored long-term memory in R6/2 mice. Thus, our results suggest that occlusion of PKA-dependent processes is one of the molecular mechanisms underlying cognitive decline in R6 animals.

Rolipram improves renal perfusion and function during sepsis in the mouse.

Microcirculatory dysfunction is correlated with increased mortality among septic patients and is believed to be a major contributor to the development of acute kidney injury (AKI). Rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, has been shown to reduce microvascular permeability and in the kidney, increase renal blood flow (RBF). This led us to investigate its potential to improve the renal microcirculation and preserve renal function during sepsis using a murine cecal ligation and puncture (CLP) model to induce sepsis. Rolipram, tested at doses of 0.3-10 mg/kg i.p., acutely restored capillary perfusion in a bell-shaped dose-response effect with 1 mg/kg being the lowest most efficacious dose. This dose also acutely increased RBF despite transiently decreasing mean arterial pressure. Rolipram also reduced renal microvascular permeability. It is noteworthy that delayed treatment with rolipram at 6 hours after CLP restored the renal microcirculation, reduced blood urea nitrogen and serum creatinine, and increased glomerular filtration rate at 18 hours. However, delayed treatment with rolipram did not reduce serum nitrate/nitrite levels, a marker of nitric oxide production, nor reactive nitrogen species generation in renal tubules. These data show that restoring the microcirculation with rolipram, even with delayed treatment, is enough to improve renal function during sepsis despite the generation of oxidants and suggest that PDE4 inhibitors should be evaluated further for their ability to treat septic-induced AKI.

Inhibition of chlorine-induced lung injury by the type 4 phosphodiesterase inhibitor rolipram.

Chlorine is a highly toxic respiratory irritant that when inhaled causes epithelial cell injury, alveolar-capillary barrier disruption, airway hyperreactivity, inflammation, and pulmonary edema. Chlorine is considered a chemical threat agent, and its release through accidental or intentional means has the potential to result in mass casualties from acute lung injury. The type 4 phosphodiesterase inhibitor rolipram was investigated as a rescue treatment for chlorine-induced lung injury. Rolipram inhibits degradation of the intracellular signaling molecule cyclic AMP. Potential beneficial effects of increased cyclic AMP levels include inhibition of pulmonary edema, inflammation, and airway hyperreactivity. Mice were exposed to chlorine (whole body exposure, 228-270 ppm for 1 h) and were treated with rolipram by intraperitoneal, intranasal, or intramuscular (either aqueous or nanoemulsion formulation) delivery starting 1h after exposure. Rolipram administered intraperitoneally or intranasally inhibited chlorine-induced pulmonary edema. Minor or no effects were observed on lavage fluid IgM (indicative of plasma protein leakage), KC (Cxcl1, neutrophil chemoattractant), and neutrophils. All routes of administration inhibited chlorine-induced airway hyperreactivity assessed 1 day after exposure. The results of the study suggest that rolipram may be an effective rescue treatment for chlorine-induced lung injury and that both systemic and targeted administration to the respiratory tract were effective routes of delivery.

Intra-abdominal use of phosphodiesterase type 4 inhibitor in postoperative adhesion prevention: an experimental study on mice.

OBJECTIVE: The study was designed to assess the value of intraperitoneal use of rolipram for adhesion prevention and to compare the results with placebo and a sodium hyaluronate/carboxymethylcellulose absorbable barrier (Seprafilm), in a murine cecal abrasion model. DESIGNS: Twenty-four Balb/c mice were subjects of this study. Intra-abdominal adhesions were created with a multiple-abrasion model consisting of meticulous abrasion of the cecum and small-bowel segments with strokes of a dental toothbrush. Animals in groups R (n = 8) received 1 mL of rolipram intraperitoneally. Seprafilm was placed over the viscera under the incision in group S animals (n = 8). Group C animals (n = 8) were reserved as control and received nothing. Animals were killed on day 22. MAIN OUTCOME MEASURES: The adhesions were evaluated with 2 different observational scoring systems, the Majuzi System and the Linsky Scale. After completion of observational evaluation, the cecum and small bowel of the animals were excised and sent to the pathology laboratory for histopathologic examination. The extent of inflammatory response, the extent of the fibrotic reaction, the extent of the necrosis and abscess formation, and the extent of foreign body reaction were histologically evaluated. RESULTS: The mean Majuzi System scores of groups R and S were similar to each other and significantly less than control group. Also, all scores of the Linsky Scale in group R were similar to those in group S, and significantly less than those in the control group. Histologically, the mean score of inflammatory response in group R was less than both those in group C and group S. The mean score of fibrotic reaction in group R was significantly less than those in the control group. CONCLUSION: These results indicate that rolipram may be an effective material in prevention of postoperative intra-abdominal adhesions, but it is obvious that further studies are needed to validate the results of this limited initial study.

Effects of repeated treatment with phosphodiesterase-4 inhibitors on cAMP signaling, hippocampal cell proliferation, and behavior in the forced-swim test.

The effects of repeated treatment with the phosphodiesterase-4 (PDE4) inhibitors rolipram, piclamilast, and 4-(2-(3-(cyclopentyloxy)-4-methoxyphenyl)-2-phenylethyl)pyridine (CDP840), which differ in their interactions with high- and low-affinity binding conformers of the enzyme, were contrasted to those of acute treatment on cAMP signaling, hippocampal cell proliferation, and immobility in the forced-swim test in rats. Repeated treatment with rolipram (1 and 3 mg/kg), piclamilast (0.3 and 1 mg/kg), or CDP840 (10 and 30 mg/kg) for 16 days increased cAMP and phosphorylation of cAMP response element binding protein (pCREB) in hippocampus and prefrontal cortex. In addition, repeated treatment with the PDE4 inhibitors increased proliferation and survival of newborn cells in the hippocampus and produced antidepressant-like effects on behavior, as evidenced by decreased immobility in the forced-swim test. Acute treatment with rolipram (3 mg/kg), piclamilast (1 mg/kg), or CDP840 (30 mg/kg) induced transient increases in cAMP and pCREB in hippocampus and prefrontal cortex, but the dose and time dependence of these effects did not parallel the behavioral effects. Compared with rolipram and piclamilast, repeated treatment with CDP840 exerted lesser effects on neural and behavioral measures, probably because of its weak interaction with the high-affinity binding conformer of PDE4. This suggests the relative importance of the high-affinity binding conformer in the mediation of the long-term effects of PDE4 inhibition on cAMP/pCREB signaling, hippocampal cell proliferation, and antidepressant-like effects on behavior.

Chemical Stimulation of Rodent and Human Cortical Synaptosomes: Implications in Neurodegeneration.

Synaptic plasticity events, including long-term potentiation (LTP), are often regarded as correlates of brain functions of memory and cognition. One of the central players in these plasticity-related phenomena is the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR). Increased levels of AMPARs on postsynaptic membranes thus constitute a biochemical measure of LTP. Isolated synaptic terminals (synaptosomes) are an excellent ex vivo tool to monitor synaptic physiology in healthy and diseased brains, particularly in human research. We herein describe three protocols for chemically-induced LTP (cLTP) in synaptosomes from both rodent and human brain tissues. Two of these chemical stimulation protocols are described for the first time in synaptosomes. A pharmacological block of synaptosomal actin dynamics confirmed the efficiency of the cLTP protocols. Furthermore, the study prototypically evaluated the deficiency of cLTP in cortical synaptosomes obtained from human cases of early-onset Alzheimer's disease (EOAD) and frontotemporal lobar degeneration (FLTD), as well as an animal model that mimics FLTD.

Phosphodiesterases 2, 3 and 4 can decrease cardiac effects of H2-histamine-receptor activation in isolated atria of transgenic mice.

Histamine exerts cAMP-dependent positive inotropic effects (PIE) and positive chronotropic effects (PCE) on isolated left and right atria, respectively, of transgenic mice which overexpress the human H2-receptor in the heart (=H2-TG). To determine whether these effects are antagonized by phosphodiesterases (PDEs), contractile studies were done in isolated left and right atrial preparations of H2-TG. The contractile effects of histamine were tested in the additional presence of the PDE-inhibitorserythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA, 1 µM, PDE2-inhibitor) or cilostamide (1 µM, PDE3-inhibitor), rolipram (10 µM, a PDE4-inhibitor), and their combinations. Cilostamide (1 µM) and EHNA (1 µM), rolipram (1 µM), and EHNA (1 µM) and the combination of rolipram (0.1 µM) and cilostamide (1 µM) each increased the potency of histamine to elevate the force of contraction (FOC) in H2-TG. Cilostamide (1 µM) and rolipram (10 µM) alone increased and EHNA (1 µM) decreased alone, and their combination increased the potency of histamine to increase the FOC in H2-TG indicating that PDE3 and PDE4 regulate the inotropic effects of histamine in H2-TG. The PDE inhibitors (EHNA, cilostamide, rolipram) alone did not alter the potency of histamine to increase the heart beat in H2-TG whereas a combination of rolipram, cilostamide, and EHNA, or of rolipram and EHNA increased the potency of histamine to act on the beating rate. In summary, the data suggest that the PCE of histamine in H2-TG atrium involves PDE 2 and 4 activities, whereas the PIE of histamine are diminished by activity of PDE 3 and 4.

Local delivery of rolipram, a phosphodiesterase-4-specific inhibitor, augments bone morphogenetic protein-induced bone formation.

Recombinant human bone morphogenetic protein (rhBMP) is a promising therapeutic cytokine for the induction of bone formation, but a weak response in humans remains a major hurdle in its therapeutic application. We have previously reported an rhBMP-2-induced increase in the bone mass of mice receiving systemic rolipram, a specific inhibitor of phosphodiesterase-4. To overcome the side effects of systemic administration of rolipram, we examined the effects of its local release. Polyethylene glycol discs were used as a delivery system. The discs were impregnated with rhBMP-2 and rolipram and implanted into the dorsal muscle pouches in mice. Bone formation was assessed by measuring the bone mineral content (BMC) of the formed bone. First, to determine the optimal dose of rolipram, we added 0-5000 nmol rolipram and 5 microg rhBMP-2 to the pellets and found that 500 nmol rolipram was the most effective concentration for inducing bone formation after 4 weeks. Second, to examine the time course of bone formation, we implanted 5 microg rhBMP-2 with 0 or 500 nmol rolipram and killed mice 5, 7, 10, 14, or 21 days after implantation. Bone formation was accelerated in the rolipram group. Finally, to determine the rolipram-induced increase in the effect of BMP, BMC obtained after treatment with 5 microg rhBMP-2 and 500 nmol rolipram was compared with that obtained after treatment with 5-9 microg rhBMP-2 without rolipram, 4 weeks after implantation. The results indicated that 500 nmol rolipram enhanced the effect of rhBMP-2 by almost 1.5-fold. In summary, locally released rolipram enhanced the capacity of rhBMP-2 to induce bone formation, an effect previously reported with systemic administration. These findings may decrease the cost and increase the efficacy of rhBMP-2 treatment.

Selective Modulation of A1 Astrocytes by Drug-Loaded Nano-Structured Gel in Spinal Cord Injury.

Astrogliosis has a very dynamic response during the progression of spinal cord injury, with beneficial or detrimental effects on recovery. It is therefore important to develop strategies to target activated astrocytes and their harmful molecular mechanisms so as to promote a protective environment to counteract the progression of the secondary injury. The challenge is to formulate an effective therapy with maximum protective effects, but reduced side effects. In this study, a functionalized nanogel-based nanovector was selectively internalized in activated mouse or human astrocytes. Rolipram, an anti-inflammatory drug, when administered by these nanovectors limited the inflammatory response in A1 astrocytes, reducing iNOS and Lcn2, which in turn reverses the toxic effect of proinflammatory astrocytes on motor neurons in vitro, showing advantages over conventionally administered anti-inflammatory therapy. When tested acutely in a spinal cord injury mouse model, it improved motor performance, but only in the early stage after injury, reducing the astrocytosis and preserving neuronal cells.

Pathophysiological effects of intravenous phosphodiesterase type 4 inhibitor in addition to surfactant lavage in meconium-injured newborn piglet lungs.

BACKGROUND: Nonsteroidal anti-inflammatory drugs, such as selective phosphodiesterase type 4 (PDE4) inhibitors have potential anti-inflammatory and respiratory smooth muscle relaxation effects. This study aimed to investigate the pathophysiological effects of an intravenous PDE4 inhibitor (rolipram) and surfactant lavage (SL) in a newborn piglet model of meconium aspiration syndrome (MAS). METHODS: MAS was induced in 25 newborn piglets, which were randomly divided into control and four SL treatment groups administered with different doses of intravenous rolipram (0, 0.1, 0.5, and 1 mg/kg). Cardiopulmonary variables were monitored and recorded. The experimental time was 4 hours. Serial blood was drawn for blood gas and biomarker analyses. Lung tissue was examined for histological analysis. RESULTS: All SL-treated groups revealed improved oxygenation during the 4-hour experiments and had significantly lower peak inspiratory pressure levels than the control group at the end of experiments. All SL plus rolipram-treated groups exhibited significantly higher lung compliance than the control group. However, the animals receiving high-dose (0.5 and 1.0 mg/kg) rolipram demonstrated significantly elevated heart rates. Lung histology of the nondependent sites revealed significantly lower lung injury scores in all SL-treated groups compared with that in the control group, but there were no differences among the rolipram-treated groups. CONCLUSIONS: In addition to SL, intravenous PDE4 inhibitors may further improve lung compliance in treating MAS; however, it is necessary to consider cardiovascular adverse effects, primarily tachycardia. Further investigations are required before the clinical application of intravenous PDE4 inhibitor as an anti-inflammatory agent to treat severe MAS.